Culture of Primary Ciliary Dyskinesia Epithelial Cells at Air-Liquid Interface Can Alter Ciliary...

Culture of Primary Ciliary Dyskinesia Epithelial Cells atAir-Liquid Interface Can Alter Ciliary Phenotype butRemains a Robust and Informative Diagnostic AidRobert A. Hirst1., Claire L. Jackson2,3., Janice L. Coles2,3, Gwyneth Williams1, Andrew Rutman1,

Patricia M. Goggin2,3, Elizabeth C. Adam2,3, Anthony Page2,3, Hazel J. Evans2, Peter M. Lackie3,

Christopher O’Callaghan1,4, Jane S. Lucas2,3*

1 Primary Ciliary Dyskinesia Centre Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, United Kingdom, 2 Primary Ciliary Dyskinesia

Centre, University Hospital Southampton, Southampton, United Kingdom, 3 Clinical and Experimental Sciences, Southampton National Institute for Health Research

Respiratory Biomedical Research Unit, University of Southampton, Southampton, United Kingdom, 4 Department of Respiratory Medicine, Institute of Child Health,

University College London & Great Ormond Street Children’s Hospital, London, United Kingdom

Abstract

Background: The diagnosis of primary ciliary dyskinesia (PCD) requires the analysis of ciliary function and ultrastructure.Diagnosis can be complicated by secondary effects on cilia such as damage during sampling, local inflammation or recentinfection. To differentiate primary from secondary abnormalities, re-analysis of cilia following culture and re-differentiationof epithelial cells at an air-liquid interface (ALI) aids the diagnosis of PCD. However changes in ciliary beat pattern of ciliafollowing epithelial cell culture has previously been described, which has brought the robustness of this method intoquestion. This is the first systematic study to evaluate ALI culture as an aid to diagnosis of PCD in the light of these concerns.

Methods: We retrospectively studied changes associated with ALI-culture in 158 subjects referred for diagnostic testing attwo PCD centres. Ciliated nasal epithelium (PCD n = 54; non-PCD n = 111) was analysed by high-speed digital videomicroscopy and transmission electron microscopy before and after culture.

Results: Ciliary function was abnormal before and after culture in all subjects with PCD; 21 PCD subjects had a combinationof static and uncoordinated twitching cilia, which became completely static following culture, a further 9 demonstrated adecreased ciliary beat frequency after culture. In subjects without PCD, secondary ciliary dyskinesia was reduced.

Conclusions: The change to ciliary phenotype in PCD samples following cell culture does not affect the diagnosis, and incertain cases can assist the ability to identify PCD cilia.

Citation: Hirst RA, Jackson CL, Coles JL, Williams G, Rutman A, et al. (2014) Culture of Primary Ciliary Dyskinesia Epithelial Cells at Air-Liquid Interface Can AlterCiliary Phenotype but Remains a Robust and Informative Diagnostic Aid. PLoS ONE 9(2): e89675. doi:10.1371/journal.pone.0089675

Editor: Shama Ahmad, University of Colorado, Denver, United States of America

Received November 27, 2013; Accepted January 21, 2014; Published February 25, 2014

Copyright: � 2014 Hirst et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: The national diagnostic PCD service in Southampton and Leicester is funded by NHS National Specialised Commissioning Team, UK.The funders had norole in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

. These authors contributed equally to this work.

Introduction

Primary ciliary dyskinesia (PCD) is a rare inherited multi-genic

disorder of cilia, impairing mucociliary clearance. It has a

prevalence of approximately 1:10,000 to 40,000 in Europe [1,2]

with a prevalence as high as 1:2,200 in certain ethnic backgrounds

[3]. Impaired function of motile cilia causes the typical signs and

symptoms of PCD. Abnormal mucociliary clearance causes wet

cough and rhinitis from soon after birth, recurrent upper and

lower respiratory infections, bronchiectasis, sinusitis and otitis

media. Approximately 50% of subjects have situs abnormalities

and subfertility is common [2–4]. To date defects in 20 genes have

been shown to cause PCD through the defective coding of proteins

that construct the ciliary axoneme or of cytoplasmic proteins that

are directly responsible for assembly of ciliary axoneme structures

[2,5].

Subjects with an indicative clinical history should be referred to

a specialist PCD service for investigations that may include nasal

nitric oxide (nNO) concentration, assessment of ciliary function

and ultrastructural axonemal defects [4]; Nasal brush biopsy

provides a sample of live and intact ciliated epithelium for

assessing ciliary function and ultrastructure. Ciliary function is

assessed by high-speed digital video microscopy (HSV) under

controlled conditions, which allows ciliary beat frequency (CBF)

and detailed beat pattern (CBP) analysis [6]. Ciliary ultrastructure

is determined by counting the percentage of structural defects seen

by transmission electron microscopy (TEM) of cilia in transverse

section [7]. Diagnosis of PCD is often difficult because referred

subjects have frequent upper respiratory tract infections with

PLOS ONE | www.plosone.org 1 February 2014 | Volume 9 | Issue 2 | e89675

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consequent inflammation and damage to the epithelium resulting

in secondary ciliary dyskinesia. The sampling itself damages the

epithelium, and secondary dyskinesia is frequently seen in

completely healthy individuals. The diagnosis is further compli-

cated by some genotypes of PCD being associated with normal

ciliary ultrastructure [8–12]. Additional methods to help resolve

difficult diagnostic cases that have been evaluated include genetic

testing [5], which can detect approximately 50% of the PCD cases,

radioaerosol mucociliary clearance [13], and immunofluorescence

microscopy [14].

Using their ability to regrow healthy ciliated epithelium from an

initial sample with a high degree of secondary damage Jorrisen et

al [15,16] first reported the use of cell culture in PCD using a

submerged method. The resulting submerged spheroidal cell

clusters rotate following ciliogenesis if ciliary motility is normal.

Assessment of CBF in suspension culture revealed that some

samples from PCD subjects had an abnormal frequency prior to

culture which became normal after ciliogenesis, but this had not

previously been described in respiratory epithelial cells cultured at

an air-liquid interface (ALI) [16]. Culturing followed by ciliation at

ALI [17,18]] has the advantage of yielding more cells than the

submerged culture technique, the cilia produced are more

numerous and can survive for up to 11 months (unpublished

data, Leicester and Southampton), allowing reanalysis of CBF,

CBP and TEM ultrastructure of the axoneme, and this has greatly

aided the diagnosis of PCD [18].

The PCD diagnostic centres in the UK [19] have found the

reanalysis of cilia following ALI culture particularly helpful for

confirming that abnormalities seen on the fresh nasal brushing are

due to PCD rather than a secondary dyskinesia. The hypothesis of

this study was ‘‘CBF of the biopsy is the same before and after

culture in a large number of PCD cases’’ This was prompted by

our observation that in some patients with PCD we observed a

change in ciliary phenotype following culture of epithelial cells. As

a consequence, this retrospective study was designed to system-

atically evaluate the ciliary function and ultrastructure of samples

before and after ALI-culture. Here we present a new, definitive

and independent observation that ALI culture can, in some cases,

cause the ciliary phenotype of PCD to become static.

Methods

Local and national R&D and ethical approvals were obtained

(Southampton and South West Hampshire Research Ethics

Committee A CHI395, 07/Q1702/109; University Hospitals of

Leicester Research Ethics Committee UHL10049, 06/Q2501/

68). All subjects gave written consent for sampling and for their

data to be used.

Subjects and samplesBetween 2009 and 2012, 158 subjects referred to centres in

Leicester and Southampton had analysis of cilia before and after

culture at ALI as part of routine PCD diagnostics. An additional 7

subjects had insufficient cilia for analysis prior to culture, but

sufficient following culture at ALI. Leicester and Southampton

PCD centres are two of the three national diagnostic centres in

England working to share protocols and standards. The HSV and

TEM data is audited between the centres to assess accuracy of

reporting and to maintain consistency of reporting across the

service.

A cytology brush (Olympus Keymed Ltd, 2mm diameter,

Southend, UK) was inserted approximately 5 cm into the patients

naris and the nasal epithelium was brushed. This method obtained

strips of nasal ciliated epithelium, and each brushing contained up

to 10,000 cells. Each sample was divided in to three to allow,

ciliary functional analysis, fixation for TEM and cell culture.

Brushings were only performed if patients had been free from an

upper respiratory tract infection for at least four weeks. HSV

analysis was conducted on all samples pre- and post- ALI culture.

TEM was conducted on all samples with any abnormality of CBP

or CBF pre-culture, but not necessarily if function was entirely

normal. TEM was generally only conducted following culture if it

was expected to aid diagnostic decision making e.g. if the pre-

culture TEM sample was insufficient for full analysis, had severe

secondary damage or provided an equivocal result.

High speed video analysis of ciliary functionProtocols were similar across the centres, but equipment

differed,(Table 1) hence baseline CBF values were considered to

be centre dependent. Cells were suspended in HEPES (20 mM)

buffered medium 199 containing penicillin (50 mg/ml), strepto-

mycin (50 mg/ml) and Fungizone (1 mg/ml). Strips of ciliated

epithelium (37uC) were imaged using an x100 objective and

digitally recorded using a high speed camera. Ciliary activity was

recorded at a rate of 500 frames per second (fps) and played back

at reduced frame rates for CBP and CBF analysis. CBF was

calculated using the following equation [CBF = (500/number of

frames for 10 ciliary beats) x 10] and a mean CBF was reported

from a minimum of 6 measurements on independent ciliated

strips. The normal range for mean CBF was 11–20 Hz in

Southampton, 10–14 Hz in Leicester. The key differences in

method between the centres for CBF measurement are that in

Leicester a glass chamber slide is used for the biopsy sample,

whereas in Southampton a plastic chamber cassette is used. The

microscopy for CBF determination is also slightly different in that

Leicester uses a conventional microscope whereas in Southampton

an inverted microscope is used. We believe that these differences

are unlikely to account for the different normal range data

between the two centres. This has been consistent over many

years, has been debated at meetings of the UK diagnostics service

[19].The UK centres have audited the data and have considered

that the differences are genuine but the reasons for the differences

have not been identified. This variability did not affect diagnostic

outcomes or general agreement on CBF. Static cilia were reported

as having a CBF of 0 Hz. In the instance of static and motile cilia

being observed together a fast Fourier transform (FFT) algorithm

was employed to measure the mean CBF in areas of ciliary beating

(in Southampton). CBP was assessed by experienced observers.

Dyskinesia was defined as an abnormal ciliary beat pattern that

included uncoordinated or asynchronous ciliary beating, reduced

beat amplitude, vibratory pattern, hyperfrequent, twitching or

static cilia.

Transmission electron microscopyCiliated epithelium for TEM was fixed in 3–4% glutaraldehyde

and prepared, imaged and analysed in a blinded fashion as

previously described [20]. The percentage of ultrastructural

defects was determined by analysis of cilia in cross section. The

number of cross sections analysed (typically 300 cilia) was dictated

by the size and secondary damage within the fixed sample.

Common defects included missing outer and inner dynein arms

alone or together, truncated outer dynein arms and microtubular

disarrangement.

Air-liquid interface cell cultureThe cell culture methodology was as previously described16. In

brief, nasal brush biopsies were grown on collagen (0.3 mg/ml,

Nutacon, Netherlands) coated tissue culture trays (12 well) in

PCD ALI-Culture


bronchial epithelial growth medium (BEGM) (BEBM including

SingleQuots, Lonza, USA) for approximately a week. Confluent

basal cells were then expanded into collagen coated flasks (25 or

75 cm2) and the BEGM replaced three times per week.

Suspensions of the non-ciliated basal cells were then seeded

(100,000–500,000 cells per well) onto collagen-coated transwell

inserts (1.2 cm2) (Costar, Corning, USA) in a 12 well culture plate

under ALI medium (1:1 BEBM:DMEM 4.5 g/L D-glucose

including SingleQuots) until confluent. The monolayer of primary

human basal epithelial cells was then exposed to an air-liquid

interface, feeding them basolaterally only with ALI-medium

supplemented with an additional 100 nM all-trans retinoic acid

(Sigma-Aldrich, UK). The medium was exchanged three times per

week and any apical surface liquid or mucus removed. Once cilia

were observed (3–6 weeks post transition to ALI), the ciliated

epithelium was recovered (by gentle scraping using a spatula) from

the transwell for analysis of ciliary function and TEM analysis of

the axonemal structure, as described above (Leicester), or a

quarter segment of the ALI culture was mounted in the chamber

slide, prior to CBF determination (x100 objective lens) by fast

fourier transformation analysis (Southampton).

The degree of ciliation per well varied between 5–50% in each

subject and the time to ciliogenesis was also variable between 3–8

weeks post ALI culture. There was no difference in the ciliogenesis

time between PCD and non-PCD cultures.

Statistical analysisCBF was determined from at least 6 separate strips of

epithelium sideways on to the observer and the mean and

confidence intervals reported for each subject. Data was normally

distributed. Paired t-tests were used to compare mean CBF before

and after culture, statistical analysis was performed in SPSS

version 20 for Windows (IBM Corp., Armonk, NY) and figures

produced in GraphPad prism 5 (GraphPad, San Diego, USA). A

p-value less than 0.05 was considered significant.

Results

ParticipantsParticipants were patients referred to the national PCD centres

in Southampton (n = 92) and Leicester (n = 73) for diagnostic

testing. We included consecutive subjects who had paired analysis

of ciliary function before and following ALI culture as part of their

PCD diagnostic assessment. Seven subjects whose samples were

insufficient for analysis but whose post-culture sample was used for

diagnostic analysis are not included in the study. Forty-one

participants from Leicester and 13 from Southampton were

diagnosed with PCD based on a combination of clinical history,

ciliary function and ultrastructure4 (Table 2). The centres share

protocols, however, equipment and subject demographics differ

and the two centres have slightly different normal ranges.

Following rigorous review across centres we have found that these

differences in CBF (but not CBP) did not alter sample processing

pathway or ultimately subject diagnosis. Data for the two centres is

therefore presented separately as well as combined (Table 3 and

Figure 1). All the non-PCD subjects in this study were referred for

PCD testing and therefore have upper and/ or lower respiratory

symptoms.

Samples from PCD subjects before and after ALI cultureThe ciliary beat pattern of samples from subjects who were

finally diagnosed with PCD was uniformly abnormal prior to

culture. Dyskinetic patterns included static cilia, static in combi-

nation with twitching cilia, cilia with rigid stroke lacking full sweep

and a rotating pattern. Beat frequency was outside the normal

range for the majority of PCD cases (Table 3 and Figure 1). All

samples were analysed by TEM analysis using samples taken prior

to culture. Based on our standard diagnostic criteria, 96% of these

had ultrastructural defects associated with PCD (Table 2; Figure

2); 18 had both inner and outer dynein arm defects, 6 had outer

dynein arm defects, 17 inner arm defects, 5 had microtubular

disorganization, 4 had transposition defects and 2 had normal

ciliary ultrastructure. One subject had an equivocal result prior to

culture.

Eight of 13 Southampton PCD samples and 13 of 41 Leicester

PCD samples demonstrated a combination of static and abnormal

motile cilia pre-culture, however post- culture at ALI went on to

exhibit completely static or near static (,1 Hz) cilia (supplemen-

tary data: video 1 a and b). An additional 9 samples with slow

ciliary beat frequency had further reduction of the frequency

following culture. All samples continued to demonstrate universal

dyskinesia after culture. The mean* ciliary beat frequency

decreased following culture (pre-ALI; mean 7.28 Hz; sd 4.37,

post-ALI: mean 4.13 Hz sd 5.15; p,0.001) (Table 3). (* Data from

one PCD subject was excluded from grouped analysis because it

was phenotypically a different variant of PCD. The cilia of this

subject were hyperfrequent and cilia appeared to vibrate with

reduced range movement prior to and following ALI-culture).

TEM results remained unchanged in all but one case where

analysis was repeated post ALI culture (Table 2). One sample from

Southampton did demonstrate a change in ciliary ultrastructure

following culture. This subject’s pre-culture functional analysis

Table 1. Equipment used at the PCD centres for high speed video microscopy.

Southampton Leicester

Specimen slide 0.5 mm coverwell imaging chamber (Sigma-Aldrich, Poole,UK) mounted onto a glass slide

Chamber created by the separation of a coverslip and aglass slide by two adjacent coverslips

Microscopy Olympus IX71 inverted microscope with modified condenser.Specimen inverted onto an x100 UPlan wide aperture oilobjective.

Leitz Diaplan conventional microscope with x100interference contrast plan apochromat objective lens.

Environmental control 37uC heated environmental chamber (Solent Scientific,Southampton, UK); anti-vibration table.

37uC heated stage of microscope; anti-vibration table(Wentworth Laboratories Ltd, Sandy, UK).

High speed video imaging andanalysis

Photron FASTCAM MC2 high speed video digital cameraand software.

IDT X4 high speed camera. AVI images analysed usingMotionPro software, IDT.

Fast Fourier transform algorithm Image J22 plugin (P. Lackie, Southampton, UK). Not used. All readings were done by a trained analystin a blinded fashion.

doi:10.1371/journal.pone.0089675.t001

PCD ALI-Culture


demonstrated a mean CBF of 8.3 Hz 6 0.6 with mixed static cilia

and cilia with abnormal motility (100% dyskinetic), which became

nearly static (,1 Hz) cilia following ALI culture. TEM analysis of

this subject’s cilia prior to culture demonstrated 60% dynein arm

defects (loss of both inner and outer dynein arms) which increased

to 98% following ALI culture.

The ultrastructural defects that were present in the subjects

whose ciliary function changed following culture were both inner

and outer dynein arm defects (n = 9), outer dynein arm defects

(n = 5), inner dynein arm defect (n = 12) and microtubular

disorganization (n = 3). The outer and inner and the outer dynein

arm defects had the least ciliary movement compared with the

inner arm and microtubular disorganisation group before culture.

The degree of motion seen in these EM phenotypes was decreased

following ALI culture and there was no individual EM defect that

was more prone to becoming static after ALI culture.

Figure 1. Ciliary beat frequency measured during high speed video analysis of samples before and following ALI culture at the twodiagnostic centres. Subjects were diagnosed with PCD or deemed non-PCD based on a portfolio of diagnostic measures.doi:10.1371/journal.pone.0089675.g001

Table 2. TEM results in subjects with and without PCD.

PCD Non-PCD

Southampton Leicester Southampton Leicester

TEM (pre ALI culture) OAD + IAD n = 5; OAD n = 2;IAD n = 1; MD n = 1; normal n = 2;equivocal* (60% of inner & outerarms missing) n = 1

OAD+IAD n = 13; OAD n = 4; IADn = 16; MD n = 4; Transpositionn = 4

N = 38. No ultrastructural changessuggestive of PCD.

N = 32 All Normal

EM (post ALI culture) No changes from pre cultureexcept previous equivocalsample, post ALI had 98% ofinner & outer arms missing.

No change from pre-culture N = 9 No ultrastructural changessuggestive of PCD.

N = 32 All normal

Key: TEM = transmission electron microscopy analysis; OAD = outer dynein arm defect; IAD = inner dynein arm defect; MD = microtubular disorganization defect;ALI = culture at air-liquid interface; PCD = primary ciliary dyskinesia.doi:10.1371/journal.pone.0089675.t002

PCD ALI-Culture


Samples from subjects without PCD, before and after ALIculture

Samples from subjects who finally had the diagnosis of PCD

excluded, demonstrated a range of functional ciliary abnormalities

when analysed immediately after nasal brushing. These included

areas of poor coordination, lack of full sweep and mucus

impedance (example of poor coordination: supplementary data

file: video 1). The mean ciliary beat frequency of most, but not all

samples was within the normal range before culture (Figure 1).

TEM analysis demonstrated a variable number of secondary

changes (e.g. disrupted membranes, compound cilia) but none had

axonemal abnormalities suggestive of PCD.

Following culture, all subjects were considered not to have PCD

on the basis of normal post-ALI culture ciliary function

(supplementary data file: video 2), combined with other diagnostic

criteria including TEM. The mean ciliary beat frequency tended

to decrease following culture in the Southampton cohort only,

(pre-ALI mean 14.21 Hz, sd 2.71; post-ALI mean 13.51 Hz sd

2.21; p = 0.02) but the size of this drop in frequency was small and

not considered physiologically relevant (mean paired difference

0.69 Hz; 95%CI 0.10 to 1.27) (Table 3). Samples from individuals

who did not have PCD did show some changes in CBF but these

were changes into the normal range. On occasions, some

individuals displayed an increased or decreased CBF outside of

the normal range that became normal following ALI-culture.

Ultrastructure was re-analysed by TEM post-culture in 41 non-

PCD samples. These continued to show no ultrastructural defects

suggestive of PCD.

Cilia Beat Pattern (CBP) and nasal Nitric OxideCBP and CBF was determined in all samples including the fresh

biopsy and post cultured cells. In non-PCD subjects the dyskinesia

was significantly reduced after cell culture (supplementary Video

S1 and S2) In Leicester 65625% were dyskinetic pre-culture and

55625% post culture. In Southampton, 85% of strips analysed

from non-PCD patient included dyskinetic cilia pre-culture but

following ALI-culture in each case over 75% of ciliated cells

demonstrated normality. The PCD subjects from both centres

displayed 100% abnormal cilia that showed a dyskinetic or static

beat pattern before and after culture (supplementary Video S3 and

S4). Nasal nitric oxide determination was performed at both

centres but only in a proportion of the subjects (due to age or

ability to do the manovre).The non-PCD subjects had a mean

nNO of 162678ppb (Leicester), 5706350ppb whereas the PCD

subjects had a significantly lower nNO of 23619ppb (Leicester),

34619ppb (Southampton).

Discussion

This is the largest study to evaluate analysis of ciliary function

and ultrastructure before and after air-liquid interface culture. It

confirms that post-culture changes in the phenotype of PCD

samples clarifies rather than confuse diagnostic status. Prior to the

study, Southampton and Leicester PCD Centres simultaneously

and independently recognised exacerbation of the functional

defects seen in some subjects with PCD following ALI culture.

This led to concerns about the validity of the technique. However,

this large study cohort of 54 PCD subjects and 111 non-PCD

subjects reassures us that the post-culture changes actually aid

diagnosis. In all cases dyskinesia associated with PCD was

unchanged or became more prominent. Secondary dyskinesia in

Figure 2. Representative TEM images: a) normal, b) outer andinner dynein arm defect c) microtubule defect d) transpositiondefect.doi:10.1371/journal.pone.0089675.g002

Table 3. Ciliary beat frequency (CBF) before and after culture at air-liquid interface in subjects with and without PCD.

CBF prior toculture (Hz)Range

CBF of ALIcultures (Hz)Range

CBF prior toALI culture (Hz)Mean (sd)

CBF of ALIcultures (Hz)Mean (sd)

Mean paireddifference(95%CI)before and during ALI P value

PCD Samples

Leicester (n = 41) 0 to 16.63 0 to 13.57 7.34 (4.82) 4.20 (4.56) 3.14 (1.84 to 4.44) ,0.001

Southampton(n = 12)$ 4.33 to 18.59 0 to 16.44 7.05 (2.48)* 3.89 (7.05)* 3.16 (–0.64 to 6.95) 0.094

Combined 0 to18.59 0 to 16.44 7.28 (4.37) 4.13 (5.15) 3.14 (1.90 to 4.39) ,0.001

Non- PCD Samples

Leicester (n = 32) 8.30 to 13.50 9.5 to 15.40 11.31 (1.42) 12.11 (1.30) –0.80 (–1.50 to –0.10) 0.027

Southampton (n = 79) 9.97 to 21.6 8.80 to 14.09 15.38 (2.18)# 14.09 (2.24)# 1.29 (0.55 to 2.01) 0.001

Combined 8.30 to 21.60 8.80 to 15.40 14.21 (2.71) 13.51 (2.21) 0.69 (0.10 to 1.27) 0.022

Measurements were made from high-speed video recordings. In samples with mixed static and dyskinetic cilia, a FFT algorithm was used to calculate mean CBF.$ The data from one Southampton PCD subject was excluded from the analysis because it was an outlier. The cilia of this subject were hyperfrequent and vibrating priorto and following ALI- culture. * = not different compared with Leicester. # = significantly different from Leicester.doi:10.1371/journal.pone.0089675.t003

PCD ALI-Culture


the non-PCD group improved. Cell culture, is a highly specialised

technique and our success rate of culturing nasal ciliated

epithelium from subjects using at an air liquid interface method

ranges from 54% from all biopsies up to 79% for biopsies with a

low level of dyskinetic cilia. This is a lower success rate than is

attainable using a simple submerged culture technique, but has the

advantage of yielding more, longer lived cilia, sufficient for post

culture ultrastructural analysis.

One of our findings was that in some subjects with PCD, the

CBF was within the normal range. This demonstrates that the

CBF alone cannot be a reliable determinant of PCD. The CBF

reading is normal in certain phenotypes of PCD and assessment of

CBP is required alongside the CBF for PCD diagnosis.

The reason for the PCD cilia movement becoming slower in

some subjects is unknown but we can speculate. Post-culture

functional changes were seen in subjects with a variety of

ultrastructural defects, including outer dynein arm defects,

combined inner and outer arm defects, inner arm defects,

transpositions, and microtubular disorganization defects. This

makes a genetic association less likely, although one could

speculate that an environmental condition was having a more

general effect on gene expression.

We considered whether the culture was preferentially selecting

or cloning cells that demonstrated static cilia, but on review of the

images, static and twitching cilia were generally co-located on cells

in the original brush biopsies. We wonder whether the absence of

any smooth muscle and nervous innervation in the artificial ALI

cultures may pay a role in this phenomenon as pharmacologically

active agents have been shown to regulate CBF [21]. We could

also speculate that secretions in the ALI are different and that

endogenous release of ciliary stimulation factors may be reduced in

PCD ALI cultures. In culture, the local reinforcement of beat

frequency and pattern due to mass flow of material along the

airways may be reduced compared to in vivo, increasing the

expression of dyskinetic patterns and possibly permitting the

survival of cells that may be lost in vivo.

This study has confirmed that reanalysis following ALI-culture

is useful where the original brush biopsy health was poor. Damage

due to infection, inflammation or trauma during sampling causes

secondary dyskinesia commonly making diagnostic status uncer-

tain. Reanalysis of newly differentiated cilia in epithelial cell

cultures in the non-PCD cohort confirmed normal ciliary

frequency and beat pattern, enabling us to confidently exclude

the diagnosis of PCD. ALI-culture has the additional benefit of

providing sufficient ciliated epithelium for TEM analysis of the

ciliary ultrastructure. TEM is an essential part of the diagnostic

testing for PCD, but our protocols require analysis of 300 healthy

cilia in cross section. From Southampton, 7 subjects had

insufficient cilia for a full analysis on the original brushing, but

we were able to complete the analysis using the ALI sample.

Without using ALI-culture, we would have needed to recall a

sizable percentage of subjects for repeat nasal brushing biopsy for

further ciliary function analysis, TEM or both.

In addition to the diagnostic benefits of this technique, ALI-

culture and submerged-culture of primary epithelial cells has

proved excellent models facilitating research into PCD [22] and

other [23,24] respiratory diseases. A limitation of this study is that

it is retrospective. The fact that it was conducted at two centres

which shared protocols but had different equipment has its

strengths and limitations.

In summary, respiratory epithelial cells obtained by nasal brush

biopsy showed phenotypic changes following culture at air-liquid

interface, including reduced CBF, which facilitated diagnosis or

exclusion of PCD. The phenotype of PCD appeared enhanced in

a number of subject samples following re-growth using ALI-

culture.

Supporting Information

Video S1 Ciliary activity before and after ALI culturefrom non-PCD subject showing secondary dyskinesia.Recorded at 500fps and played at 30fps.

(MPG)

Video S2 The same subjects cilia as in Video S1 withimprovement of secondary dyskinesia following culture.The image was recorded at 500fps and played back at 30fps.

(MPG)

Video S3 High speed video images of ciliary functionfrom a subject with PCD showing abnormal movement.Recorded at 500fps and played at 30fps.

(MPG)

Video S4 The same PCD subject as in Video S3 showingall of the cilia are static following culture. Recorded at

500fps and played at 30fps.

(MPG)

Acknowledgments

Sample collection was supported by the Southampton NIHR Wellcome

Trust Clinical Research Facility and NIHR Southampton Respiratory

Biomedical Research Unit. We are grateful to Amanda Harris, Karen Lock

and Woolf Walker for subject recruitment and taking clinical samples.

Author Contributions

Conceived and designed the experiments: JSL CLJ RAH. Performed the

experiments: CLJ JLC RAH ECA PMG AR GW AP. Analyzed the data:

JSL CLJ RAH. Contributed reagents/materials/analysis tools: JSL HE

CO PML. Wrote the paper: JSL RH CLJ. Responsible for HSV analysis

and ALI culture in Southampton: CLJ JLC EA. Responsible for HSV and

ALI culture in Leicester: RAH GW. Conducted EM analysis: PMG AP

AR. Developed the FFT algorithm with Image J22 software used in

Southampton: PML. Clinical leads for the diagnostic service: CO HE JSL.

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PCD ALI-Culture


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